During the past grant period(2 years) the applicant has isolated the NADH dehydrogenase complex of Paracoccus denitrificans and characterized it with respect to enzymic activities, polypeptide composition, content of non-heme iron and labile sulfide, and the nature of flavin. he has also shown that dicyclohexylcarbodiimide inhibits the NADH-Q reductase activities of those organisms which contain an energy coupling site 1 (mammals, P. denitrificans, E. coli, T. thermophilus) but not NADH-Q reductase activities of those that lack coupling site 1 (S. cerevisiae, B. subtilis). He has further shown in mammalian complex I that DCCD inhibition of NADH-Q reductase is correlated with labeling of a polypeptide of Mr=29,000 with [14C]DCCD. Studies planned for this grant period are as follows: (I) Resolution of the isolated NADH dehydrogenase complex of Paracoccus by detergents and/or strong chaotropes and assignment of redox centers (FMN, four FeS clusters) to subunits and determination of substrate- binding subunits. (II) Reconstitution of the NADH dehydrogenase complex from the resolved fragments (or subunits) and determination of the minimum number of subunits required for reconstitution of electron transfer and proton translocation and for interaction of the complex with the bc1 complex. (III) Reconstitution of rotenone-sensitivity and H+ translocation function of NADH dehydrogenase complex from the isolated NADH dehydrogenase complex + phospholipids, the be1 complex of Paracoccus, and/or polypeptide that might have been separated during purification. (IV) Study of the sequence of electron carriers in NADH dehydrogenase complex, using chemical modification techniques to achieve interruption of electron transfer at various points. (V) Determination and characterization of the DCCD binding subunit in the NADH dehydrogenase complex of Parcoccus and other organisms that contain energy coupling site 1. (VI) Isolation of rotenone-insensitive and Q- deficient mutants. Study of the relationships among energy coupling site 1, FeS cluster N2 and rotenone sensitivity, using rotenone-sensitive membranes and rotenone-insensitive mutants. Study of the role of ubiquinone-10 in electron transfer and proton translocation at energy coupling site 1 with the use of Q-deficient mutants.